The dopamine transporter has been a primary molecular target for the development of potential cocaine abuse therapeutics. Although the inhibition of dopamine reuptake is likely to be an oversimplified explanation for the addictive properties of cocaine, it remains the foremost mechanistic candidate responsible for the reinforcing effects of this drug of abuse. The development of novel probes for the dopamine transporter that are structurally dissimilar but retain neurochemical equivalence to cocaine is the focus of our research program. We are particularly interested in those compounds that bind to the dopamine transporter with equal or higher affinity than cocaine but are not cocaine-like in animal models of drug abuse. These compounds will help characterize binding domains on the dopamine transporter and allow the determination of functional correlates to those sites. Ultimately, it is envisioned that a compound may be developed that binds to the dopamine transporter and prevents cocaine from binding without having the addiction liability of cocaine itself. We have explored the design and synthesis of novel analogs based on two classes of dopamine uptake inhibitors and have evaluated them in vitro (radioligand binding assays and inhibition of [3H]dopamine uptake) and in vivo (locomotor activity in mice, drug discrimination in rats and self administration studies in monkeys). The first class of compounds are based on 3-alpha-(diphenylmethoxytropane (benztropine). Previous chemical modification at the 3-position of this molecule allowed the discovery of a potent class of dopamine uptake inhibitors that retained high affinity binding at muscarinic receptors but were highly selective over serotonin and norepinephrine transporters. These compounds typically blocked dopamine reuptake, as does cocaine, but were not efficacious as locomotor stimulants and were not cocaine-like in either drug discrimination or self administration studies. In an attempt to eliminate the muscarinic antagonist properties of these compounds, a large series of N-substituted 4',4@-difluoro analogs were prepared. Many of these compounds retained high affinity binding at the dopamine transporter but demonstrated a significant decrease in affinity for muscarinic receptors. Behavioral evaluation of these compounds, as with the previous series did not demonstrate a cocaine-like behavioral profile. Further, several of the N-substituted analogs did not potentiate the effects of cocaine, as most other dopamine uptake inhibitors and muscarinic antagonists have been shown to do. Pharmacokinetic analysis as well as chronic studies are currently underway to further characterize the pharmacology of these compounds and their potential as pharmacotherapeutics. In addition, an 125I- azido- derivative of the N-butylphenyl substituted 4',4@-difluoro benztropine has been prepared and shown to irreversibly interact with the cloned dopamine transporter. This photoaffinity label will be used in future studies to determine the transmembrane helices at which this class of compounds binds and compare the binding domains with those labeled by photoaffinity ligands based on cocaine and GBR 12909. Numerous chemical modifications have been made to a second class of compounds, based on rimcazole, a dopamine uptake inhibitor/sigma ligand that attenuates the locomotor activity induced by cocaine. Substitutions at the carbazole ring system as well as at the terminal piperazine nitrogen have been made. Thusfar, structure-activity relationships suggest that the dopamine transporter is very sensitive to structural changes in this molecule, with most modifications resulting in a decrease in binding affinity. However, several analogs were discovered to have equal or slightly higher affinity at the dopamine transporter than rimcazole (Ki=60-260 nM). These compounds have also been evaluated for their binding affinities at sigma 1 and 2 receptors. Selected compounds have been evaluated behaviorally and so far, like the parent rimcazole they fail to demonstrate a cocaine- like behavioral profile. Cocaine-interaction studies are underway. In addition, an irreversible ligand based on rimcazole has been discovered and promises to be an important research tool with which to characterize high and low affinity sites on the dopamine transporter .